This invention relates to the field of blood coagulation testing, specifically to reagents useful in measuring various coagulation factors and a method for the preparation of such reagents.
Effective medical treatment of certain patients requires the measurement of the blood's clotting ability. In general, the blood coagulation process depends upon the presence of interactive blood components which cause blood to form a gel. The first stage of coagulation is formation of blood thromboplastin. Several intrinsic blood constituents interact to form blood thromboplastin, one of which is platelets. This is followed by the conversion of prothrombin to thrombin, a process aided by the now present blood thromboplastin. Third, thrombin acts on fibrinogen present in the blood to form fibrin, an insoluble plasma protein. Fibrin, by its insoluble nature, forms a clot. Depending upon the site of clotting, step one above may be obviated. For example, tissue thromboplastin is continuously present in nearly all body tissues and is also active in prothrombin conversion.
If any one of the factors essential to clotting is absent, or present in insufficient quantity, the clotting ability of blood is seriously impaired. The time for the clotting process to take place is measured as an indicator. The clinician, once armed with the knowledge that the patient cannot form clots in normal time, can adjust his treatment program accordingly. Postoperatively, healing may be hindered by low clotting time. Therefore, postoperative treatment may be specially tailored once low clotting time is realized. As a diagnostic tool, absence or low incidence of a factor necessary to clotting is useful in determining a patient's disease. In this regard, a factor sensitive reagent is highly desirable.
In a typical test procedure, blood is withdrawn from the human body and centrifuged to remove the platelets and blood cells. The supernatant plasma is withdrawn and mixed with a partial thromboplastin time reagent containing an activator and a platelet substitute. After a period the clotting factors are activated. After the addition of calcium, the solution is put into a cuvette and placed in a spectrophotometer for measuring the change in absorbency. Alternatively formation of a clot is observed directly by the human eye.
There are a variety of commercially available reagents for measuring partial thromboplastin. It is known that certain ellagic acid solutions are successful contact activators. Thus, ellagic acid forms an effective activated cephaloplastin reagent. Several formulations utilizing ellagic acid, generally at 100 micromolar concentration, are commercially available, for example, Actin* (available from American Dade, division of Baxter Travenol Diagnostics, Inc. of Miami, Fla.). These commercially available cephaloplastin reagents also include a phospholipid, typically a cephalin, as a platelet substitute. Further, some of these reagents contain metal ions at various concentration ratios to ellagic acid. The metal ion concentration ratios from some of these activated partial thromboplastin time reagents have been determined using atomic absorbtion spectroscopy. The following table summarizes this data:
______________________________________ Activated Ion Thrombofax Actin Actin-FS ______________________________________ Ca.sup.2+ .09* .44 1.21 Co.sup.2+ &lt;0.01 &lt;0.02 &lt;0.02 Cu.sup.2+ &lt;0.03 &lt;0.02 &lt;0.02 Fe.sup.2+ &lt;0.03 &lt;0.03 &lt;0.03 Mg.sup.2+ 0.25 0.30 0.49 Mn.sup.2+ &lt;0.01 &lt;0.01 &lt;0.01 Sr.sup.2+ &lt;0.05 &lt;0.03 &lt;0.03 Zn.sup.2+ 0.05 0.01 &lt;0.01 Total &lt;0.53 &lt;0.86 &lt;1.82 M.sup.2 ______________________________________ Thrombofax is the registered trademark of Ortho Diagnostics, Raritan, NJ. Actin and ActinFS are the registered trademarks of American Dade, Miami, FL. *Metal ion to ellagic acid molar concentration ratio. Ellagic acid concentration at 100 micromolar.
U.S. Pat. No. 4,732,860 to Bartl teaches a process using ellagic acid, preferably present at between 1.3 micromolar and 1.5 micromolar in combination with a phospholipid, including a cephalin to determine prekallikrein content and partial thromboplastin time. When these values are to be determined simultaneously, calcium ions are an essential ingredient in the process as they are integrally involved in the clotting process. Preferable calcium concentrations are taught to be 25,000 to 40,000 micromolar but may range from 1000 micromolar to 100,000 micromolar, some 769 to 76,923 times the ellagic acid concentration. When used, calcium ion is subsequent to the addition of a first reagent to the plasma sample.
The difficulty encountered with certain presently commercially available partial thromboplastin time reagents (APTT reagents) is that they often take a long time to prepare, a manufacturing drawback which increases expense. Further, these ellagic acid suspensions are not stable. With time ellagic acid may precipitate from the solution so that such reagents must be shaken before use. More preparation time is then required and the technician must be alert to the presence of precipitated ellagate compounds. As a result, the APTT test using these reagents is often unreproducible over a prolonged period of time. Another disadvantage in these presently available reagents is that they are typically not broadly factor selective. Although several factor sensitive reagents are available, the range of factors independently measured in narrow.
U.S. Pat. No. 3,486,981 to Speck teaches a partial thromboplastin time test reagent consisting of ellagic acid as the chemical activator of the Hageman factor. This patent contains no suggestion of the importance of divalent metal ions in promoting ellagic acid activity.
A paper by Bock et al, Activation of Intrinsic Blood Coagulation by Ellagic Acid: Insoluble Ellagic Acid-Metal Ion Complexes Are the Activating Species, Biochemistry (1981), 20, 7258-7266, teaches the importance of divalent metal ions, most significantly copper, in ellagic acid activation. This article teaches that soluble ellagic acid does not initiate blood coagulation. Instead insoluble ellagate:metal ion complexes are responsible for such activity. The authors conclude that previously reported ellagic acid activity as a factor in blood coagulation was based on the presence of adventitious metal ions most likely introduced into those solutions in the diluting buffer. Concentrations of ellagic acid investigated for procoagulant activity in the article were 30 micromolar more or less. Higher concentrations of ellagic acid were required in absolute solubility testing but not for efficiency testing. The authors demonstrated ellagic acid activity at these low ellagic acid concentrations upon addition of certain metal ions, namely Cu.sup.2+, Zn.sup.2+, Co.sup.2+, and Fe.sup.3+. The authors also used calcium and magnesium but found that these two ions demonstrated weaker affinity for ellagic acid and thus, according to the authors, were not preferred as ellagic acid activators. Further, the procedure for ellagic acid preparation resulted in precipitation after overnight incubation at concentrations as low as 20 micromolar ellagic acid in the presence of divalent metal ions. Metal ion concentration ratios were 1.5 or less relative to ellagic acid.
None of these references teaches the fundamental principle of the present invention: a stable ellagic acid:cephalin:metal ion suspension wherein the metal ion species are present at molar ratios between 3 and 30 relative to the ellagic acid concentration. Further, none of these references teaches a stable reagent capable of forming a procoagulation reagent upon exposure to a source of cephalin containing ellagic acid at concentrations greater than 30 micromolar where divalent metal ion species are present at molar ratios of about 3.0 or less relative to the ellagic acid concentration.